KR101110396B1 - Method of detecting variation and kit to be used therein - Google Patents

Abstract

The present invention provides a method for detecting variation with excellent detection sensitivity using Tm analysis. A detection probe and a non-mutation probe comprising a polynucleotide complementary to a detection target sequence comprising the mutated detection site, in a sample containing a detection target DNA mutated and a non-detection DNA mutated undetected. The inhibitory polynucleotide complementary to the non-detection target sequence including the detected detection site is added to hybridize the detection probe to the DNA. Then, the hybrid formation of the DNA and the detection probe is heated to measure the fluctuation of the signal due to the temperature rise, and the fluctuation is determined by analyzing the fluctuation of the signal to determine the Tm value.

Description

Mutation detection method and kit for use {METHOD OF DETECTING VARIATION AND KIT TO BE USED THEREIN}

The present invention relates to a method for detecting mutations and a kit used therein.

As a method of interpreting the cause of all diseases, disease reversibility (easiness of disease) among individuals, and differences in drug efficacy among individuals, the detection of point mutations, so-called monobasic polymorphisms (SNPs), is widely used. It is done.

As a general detection method of point mutations, for example, (1) a direct sequencing method for amplifying a region corresponding to a detection target sequence to a target DNA of a sample and analyzing the base sequence of the amplification product obtained, (2) a pyrosequencing method, ( 3) Denaturing HPLC method which amplifies the area | region corresponding to a detection target sequence, performs HPLC in a temperature gradient column, and detects the presence or absence of a change with time eluted, and (4) the target mutation. Invadar method for detecting a mutation by detecting the fluorescence using a fluorescence probe when the fluorescent probe binds to the region to be detected, (5) PCR using a primer in which the target mutation is located in the 3 'terminal region, The ASP-PCR method which judges a variation with or without amplification is mentioned.

However, the methods of (1), (2), and (4) have low sensitivity of about 20%, about 5%, and about 5%, respectively, and the operation takes a lot of trouble and time. The method of (3) has a problem that the sensitivity is low, about 10%, and that the presence or absence of a variation can be confirmed, and that any variation cannot be interpreted in which part, and the specificity is insufficient. Moreover, the method of said (5) has a problem that although it is high in sensitivity, it is low in specificity and it is easy to produce false positives. The smaller the numerical value (%), the higher the sensitivity.

Recently, detection using Tm analysis has been performed as a detection method for point mutations. This method can be performed as follows, for example. First, using a probe complementary to a detection target sequence containing a point mutation for detection, a target single-stranded DNA in a sample and a hybrid (double-stranded DNA) of the probe are formed. Subsequently, heat processing is performed to this hybrid formation body, and the dissociation (fusion) of the hybrid accompanying a temperature rise is detected by signal measurement, such as absorbance. Then, the presence or absence of a point mutation is determined by determining the Tm value based on this detection result. The higher the homology of the hybrid body is, the higher the Tm value is, and the lower the homology is. For this reason, the Tm value (evaluation reference value) is calculated | required beforehand about the hybrid body of the detection object sequence containing a point mutation, and the probe complementary to it, and the Tm value (measurement value) of a target single-stranded DNA and the said probe is calculated previously. If it measures, the following judgments are possible. If the measurement is equal to the evaluation criteria, it can be determined that there is a match, that is, a point mutation is present in the target DNA. On the other hand, if the measurement is lower than the evaluation criteria, it can be determined that there is no mismatch, that is, no point mutation in the target DNA.

However, the detection method using such a Tm analysis has a problem that the sensitivity is low. As a specific example, there is a problem when detecting a point mutation on DNA derived from blood cells of a leukemia patient (Patent Document 1). Leukemia is a disease caused by cancer of hematopoietic stem cells in bone marrow. Among them, chronic myeloid leukemia (CML) is known as a cause of the onset of the bcr-abl1 fusion gene formed by the translocation of the chromosome 9 and the chromosome 22, and for the treatment, imatinib (abl1 kinase inhibitor) imatinib) is widely used. However, when a point mutation exists in this abl1 gene (including the abl1 gene in the said fusion gene), there exists a problem of expressing resistance to imatinib. In that case, for example, an increase in imatinib dosage, a change to another therapeutic drug, a switch to bone marrow transplantation, or the like will be required. Therefore, in the treatment of leukemia, especially CML, it is very important to detect the presence or absence of point mutations in the abl1 gene. However, even in the blood of a single CML patient, the blood cells contain a point mutation (a sequence to be detected) and a non-detection sequence (a sequence to be detected) in the abl1 gene, and the difference between the points is a point mutation. That is only a single base sequence. If so, the probe for detecting the point mutation hybridizes (matches) to the detection target sequence including the point mutation, and also hybridizes (mismatches) to the non-detection target sequence not containing the point mutation. Done. In such a case, when a melting curve showing the relationship between the signal intensity and the temperature is generated according to the Tm analysis, the peak at the high temperature side for the matching detection target sequence is the peak at the low temperature side for the non-detection sequence which is a mismatch. It becomes difficult to detect by the presence of, and a decrease in detection sensitivity is caused.

[Patent Document 1] Japanese Patent Publication No. 2004-537992

Challenges to Invent

Then, an object of this invention is to provide the detection method of the variation excellent in the detection sensitivity using Tm analysis, and the detection probe kit used for it.

Means to solve the problem

In order to achieve the above object, the method for detecting a mutation of the present invention is a sample containing a DNA to be detected in which a detection site is mutated and a non-detection DNA in which the detection site is mutated,

It is characterized by including the following (A)-(E) process.

(A) A step of adding a detection probe consisting of a polynucleotide complementary to a detection target sequence and a polynucleotide complementary to a non-detection target sequence (hereinafter referred to as an "inhibition polynucleotide") to a sample containing the DNA. As

The detection subject sequence includes the mutated detection site as the detection target DNA or a partial sequence thereof,

Wherein the non-detection target sequence includes the undetected detection site as the non-detection DNA or a partial sequence thereof,

(B) hybridizing the detection probe to the DNA;

(C) measuring the fluctuation of the signal according to the temperature change with respect to the hybrid formation of the DNA and the detection probe,

(D) analyzing the fluctuation of the signal to determine the Tm value,

(E) Process of determining presence or absence of the mutation in the said detection object site | part from the said Tm value.

The detection probe kit of the present invention is a detection probe kit used in the method for detecting mutations of the present invention.

A detection probe comprising a polynucleotide complementary to a detection target sequence, and a polynucleotide for inhibition complementary to a non-detection sequence,

The detection target sequence is a detection target DNA or a partial sequence of which the detection site is mutated, and includes the mutated detection site,

The non-detection target sequence is characterized in that the non-detection target DNA or its partial sequence unmodified the detection site includes the undetected detection site.

[Effects of the Invention]

In the method for detecting the mutation of the present invention, not only the detection probe for the detection target sequence in which the detection site is mutated, but also the polynucleotide for inhibition for the non-detection sequence in which the detection site is mutated is added to the sample. Doing. For this reason, hybridization of the said detection probe to the non-detection target sequence can be suppressed, and as a result, the mutation in the said detection site | part can be detected with the sensitivity (about 3%) superior to the past. As described above, the hybridization of the detection probe to the non-detection target sequence can be suppressed because the homology of the inhibitory polynucleotide to the non-detection sequence is higher than that of the detection probe. . Therefore, the detection method of the present invention is useful for, for example, a sample in which both the DNA to be detected and the non-detection DNA are contained in the sample. In particular, it is useful for a sample of leukemia patients, and particularly, for detecting a mutation of the abl1 gene (including a mutation of the abl1 gene in the bcr-abl1 fusion gene) in patients with chronic myelogenous leukemia (CML). . As described above, since the mutation can be detected with high sensitivity, for example, the titration of leukemia therapeutic drugs for each patient can be analyzed at the genetic level, which is a very useful method in the medical field. In addition, by using the detection probe kit of the present invention, the method for detecting the mutation of the present invention can be easily performed.

BRIEF DESCRIPTION OF THE DRAWINGS It is a graph which shows the result of Tm analysis which added the polynucleotide for inhibition in Example 1 of this invention.

FIG. 2 is a graph showing the results of Tm analysis in the absence of inhibitory polynucleotide in Comparative Example 1. FIG.

FIG. 3 is a graph showing the result of Tm analysis in which the addition ratio of the detection probe is changed in Example 1 of the present invention. FIG.

It is a graph which shows the result of Tm analysis which changed the addition ratio of the inhibiting polynucleotide in Example 1 of this invention.

It is a graph which shows the result of Tm analysis which added the polynucleotide for inhibition in Example 2 of this invention.

It is a graph which shows the result of Tm analysis which added the polynucleotide for inhibition in Example 3 of this invention.

It is a graph which shows the result of Tm analysis which added the polynucleotide for inhibition in Example 4 of this invention.

Best Mode for Carrying Out the Invention

As described above, the method for detecting a mutation of the present invention is a method for detecting a mutation of DNA in a sample, wherein the sample includes a DNA to be detected in which a detection site is mutated and a non-detection DNA in which the detection site is mutated. It is a sample containing, It is characterized by including the following (A)-(E) process.

(A) adding a detection probe consisting of a polynucleotide complementary to a detection target sequence and a polynucleotide for inhibition complementary to a non-detection target sequence, to the sample containing the DNA,

(B) hybridizing the detection probe to the DNA;

(C) measuring the fluctuation of the signal due to the temperature change with respect to the hybrid formation of the DNA and the detection probe,

(D) analyzing the fluctuation of the signal to determine the Tm value,

(E) Process of determining presence or absence of the mutation in the said detection object site | part from the said Tm value.

In the said (A) process, the said detection object sequence is a sequence containing the said detection object site | part which was mutated as said detection object DNA or its partial sequence. In addition, the said non-detection object sequence is a sequence containing the said undetected said detection object site | part as the said non-detection DNA or its partial sequence.

In the present invention, the above-described detection target sequence in which mutations exist in the detection site is referred to as "variation sequence", and the detection target DNA containing the detection target sequence is also referred to as "mutation DNA", and the above ratio in which no mutation exists in the detection site. DNA containing a detection target sequence as "normal sequence" and the said non-detection target sequence is also called "normal DNA." In addition, the variation in a detection site is also called "the variation for detection purposes." DNA in the target sample which detects the presence or absence of a mutation is also called "target DNA." Examples of the mutation to be detected in the present invention include monobasic polymorphism (SNP).

In the present invention, the DNA in the sample may be single-stranded DNA or double-stranded DNA. When the said DNA is double-stranded DNA, it is preferable to include the process of dissociating the double-stranded DNA in the said sample by heating, for example, prior to the said (B) hybridization process. By dissociating double-stranded DNA into single-stranded DNA, hybridization of the detection probe and the inhibitory polynucleotide can be efficiently performed in the next hybridization step (B).

In the present invention, the DNA in the sample may be, for example, a gene, or may be a partial sequence of the gene. The DNA in the sample may be, for example, DNA originally contained in a sample such as a biological sample. However, since the detection accuracy can be improved, for example, the amplification product amplified by the gene amplification method is preferable. Specifically, for example, a reverse transcription reaction is carried out from an amplification product amplified by a gene amplification method using DNA originally contained in the sample or RNA (total RNA, mRNA, etc.) originally contained in the sample. And amplification products amplified by gene amplification using cDNA generated by RT-PCR (Reverse Transcription PCR) as a template. The length of the amplification product is not particularly limited, but is, for example, 50 to 1000 mer, preferably 80 to 200 mer.

The sample to which the detection method of this invention is applied is not specifically limited. The present invention is, for example, as described above, for a sample containing both a DNA having a target mutation (a DNA to be detected) and a DNA having no target mutation (a non-detected DNA), as a target DNA. useful. The origin of the said DNA and RNA is not restrict | limited, For example, cells, such as various cancer cells, a virus, mitochondria, etc. are mentioned. In particular, as described above, when the mutation is detected in a biological sample (for example, a blood sample) of a leukemia patient, the darkened blood cells have a cell having a DNA having a mutation and a DNA having no mutation. Since the cells are included, the above problems are likely to occur. Therefore, the detection method of the present invention is particularly preferably applied to a sample having a DNA having a mutation and a DNA having no mutation, and applied to, for example, a biological sample of leukemia, a blood sample or a leukocyte cell as a specific example. It is preferable. In addition, in this invention, the sampling method, the DNA preparation method, etc. are not restrict | limited, A conventionally well-known method can be employ | adopted.

The variation of the detection object in the present invention is not limited. As described above, when detecting a mutation related to leukemia, it is known that the detection probe hybridizes to an undetected sequence. Therefore, as an embodiment, the method of the present invention is useful when detecting a mutation of a gene related to leukemia. As a mutation of the gene related to the said leukemia, the mutation of the abl1 gene (including the mutation of the abl1 gene in a bcr-abl1 fusion gene) is mentioned, for example. For example, in the cDNA sequence (mRNA sequence) of the abl1 gene shown in SEQ ID NO: 1, the 756th base G, the 758th base A, and the 763th base G may be mentioned. As for these bases, the following mutations have been reported, for example. In addition, the sequence of the abl1 gene is registered under NCBI Accession No. NM_005157.

G756C 756th base G mutated to C

A758T 758th base A is mutated to T

G763A The 763th base G is mutated to A

In the present invention, the detection probe may be a sequence complementary to the detection target sequence in which the detection site is mutated, and the inhibition polynucleotide may be a sequence complementary to the non-detection target sequence in which the detection site is unmuted. The length of the probe and the inhibitory polynucleotide is not particularly limited, but is preferably the same length. The sequence of the detection probe and the sequence of the inhibitory polynucleotide are 90% to excluding a site (base) that is paired with the detection site (site where the target mutation occurs) when forming a hybrid, for example. It is preferable that it is 100% identical sequence, Especially preferably, it is 100%. The detection probe and the inhibitory polynucleotide may be designed to hybridize to any of the forward and reverse chains of DNA as long as they are the same chain.

Below, the combination of the detection probe and inhibition polynucleotide used for detection of said three types of mutations (G756C, A758T, G763A) of the abl1 gene is illustrated. In each sequence, bases indicated by capital letters correspond to the 756th, 758th, and 763th positions of the abl1 gene, respectively. The present invention is not limited to these.

transition G756C ( Pure  For detection)

Detection probe sequence number 2

5'-ccgtaGtggcccccgc-3 '

Polynucleotide SEQ ID NO: 3 for Inhibition

5'-ccgtaCtggcccccgc-3 '

Variation A758T ( Pure  For detection)

Detection probe sequence number 4

5'-ccgAactggcccccgc-3 '

Polynucleotide SEQ ID NO: 5 for Inhibition

5'-cctccccgTactggcccccg-3 '

Polynucleotide SEQ ID NO: 6 for Inhibition

5'-ccg Tactggcccccgc-3 '

Variant G763A (for detecting reverse chain)

Detection probe SEQ ID NO: 7

5'-ccagtacgggAaggtgt-3 '

Inhibition Polynucleotide SEQ ID NO: 8

5'-ccagtacgggGaggtgt-3 '

In the present invention, the addition ratio of the inhibitory polynucleotide is not particularly limited, but can be appropriately determined depending on the conditions of the detection system, such as the length of the detection probe, the GC content of the detection target sequence, and the like. Although the addition ratio with respect to the said detection probe is not restrict | limited, For example, a minimum is 0.1 time or more in molar ratio, Preferably it is 1 time or more, More preferably, it is 2 times or more. In addition, an upper limit is 100 times or less in molar ratio, for example. The length of the inhibitory polynucleotide is not particularly limited and is, for example, 5 to 50 mer, preferably 10 to 30 mer, and preferably set to the same length as the detection probe.

In the present invention, the addition ratio of the detection probe complementary to the detection target sequence is not particularly limited. For example, since the detection signal can be sufficiently secured, it is preferably one or less times the molar ratio with respect to the DNA in the sample. . In addition to the addition of the inhibitory polynucleotide, by further controlling the addition ratio of the detection probe, it is possible to further improve the detection sensitivity, for example. In addition, since it is enough to just set the addition ratio of the said detection probe, operation is very simple. The addition ratio of the said detection probe is more preferably 0.1 times or less in molar ratio with respect to the said DNA. In this case, the DNA in the sample may be, for example, the sum of the DNA to be detected and the non-detection DNA, or the sum of the amplification product including the detection target sequence and the amplification product including the non-detection sequence. In addition, although the ratio of DNA to be detected in the DNA in a sample is usually unknown, as a result, the addition ratio of the said detection probe is molar ratio, for example with respect to DNA to be detected (or an amplification product containing a sequence to be detected). It is preferable to become 10 times or less, More preferably, it is 5 times or less, More preferably, it is 3 times or less. The lower limit thereof is not particularly limited. For example, the molar ratio of the detection target DNA and the like is preferably 0.001 times or more, more preferably 0.01 times or more, and more preferably 0.1 times or more.

The addition ratio of the detection probe to the DNA may be, for example, a molar ratio to double-stranded DNA or a molar ratio to single-stranded DNA. In addition, the length of the said detection probe is not specifically limited, For example, it is 5-50 mer, Preferably it is 10-30 mer.

The Tm value will be described. When the solution containing the double-stranded DNA is heated, the absorbance at 260 nm increases. This is because hydrogen bonds between both chains in double-stranded DNA are released by heating, and dissociate into single-stranded DNA (fusion of DNA). When all the double-stranded DNAs dissociate to form single-stranded DNAs, the absorbance represents about 1.5 times the absorbance at the start of heating (absorbance only of the double-stranded DNA), and thus it can be judged that the fusion is completed. Based on this phenomenon, the melting temperature Tm is generally defined as the temperature at which the absorbance reaches 50% of the total increase in absorbance.

In the present invention, the measurement of the signal fluctuation caused by the temperature rise for determining the Tm value can be performed by absorbance measurement at 260 nm, for example, based on the principle described above. More preferably, signal variation is measured using a probe labeled with a labeling substance as the detection probe. The labeling site is not particularly limited in the detection probe. In addition, the labeling substance is not particularly limited, and can usually bind to the phosphate group of the nucleotide.

As said labeling probe, the labeling probe which shows a signal by itself and does not show a signal by hybrid formation, or the labeling probe which does not show a signal by itself and shows a signal by hybrid formation is mentioned, for example. If the probe is the same as the former, the signal is not displayed when a hybrid (double-stranded DNA) is formed with the sequence to be detected, and the signal is displayed when the probe is favored by heating. In the latter probe, a signal is obtained by forming a hybrid (double-stranded DNA) with the sequence to be detected, and the signal decreases (dissipates) when the probe is favored by heating. Thus, for example, by detecting a signal by the labeling substance under signal-specific conditions (absorbance, etc.), the progress of melting and the Tm value can be determined in the same manner as in the absorbance measurement at 260 nm.

Examples of the labeling substance include, but are not limited to, fluorescent dyes (fluorophores). As a specific example of the labeling probe, for example, a probe that is labeled with a fluorescent dye and exhibits fluorescence alone and whose fluorescence is reduced by hybrid formation (for example, quenching) is preferable. A probe using such a quenching phenomenon is called a fluorescence quenching probe. Especially, as a detection probe, it is preferable that the 3 'end or 5' end of an oligonucleotide is labeled with the fluorescent dye, and it is preferable that the base of the said terminal to be labeled is C. In this case, the detection target DNA hybridized by the detection probe, so that the base paired with the terminal base C of the detection probe or a base 1 to 3 bases away from the paired base becomes G. It is preferable to design the base sequence of the probe. Such probes are generally called guanine quenching probes and are known as QProbe (registered trademark). When such guanine quenching probe hybridizes to the DNA to be detected, the terminal C labeled with the fluorescent dye approaches G in the DNA to be detected so that light emission of the fluorescent dye is weakened (fluorescence intensity decreases). Indicates a phenomenon.

Although it does not restrict | limit especially as said fluorescent dye, For example, a fluorescein, a phosphor, rhodamine, a polymethine pigment derivative, etc. are mentioned. Examples of commercially available fluorescent dyes include BODIPYFL (trade name, manufactured by Molecular Probes), FluorePrime (trade name, manufactured by Amersham Pharmacia), Fluoredite (trade name, manufactured by Mirpoa), and FAM (ABI). Company), Cy3, Cy5 (made by Amarsham Pharmacia), TARMA (made by a molecular probe company), etc. are mentioned. The detection conditions are not particularly limited and can be appropriately determined depending on the fluorescent dye to be used. As a specific example, Pacific Blue can detect, for example, the detection wavelength of 450-480 nm, TAMRA can detect, for example, the detection wavelength of 585-700 nm, and BODIPY FL, for example at the detection wavelength of 515-555 nm. By using such a probe, hybridization and dissociation can be easily confirmed by the change of a signal. On the other hand, the inhibitory polynucleotide is preferably not labeled.

Next, the detection method of this invention is demonstrated using the 758th base A point mutation (A-> T) in the abl1 gene (SEQ ID NO: 1) as an example. In addition, the present invention is characterized in that a polynucleotide for inhibition is added, and there are no limitations on other processes and conditions. The detection target sequence hybridized by the detection probe may be, for example, a full-length sequence in which the 758th base A is T-transformed in the nucleotide sequence of SEQ ID NO: 1, but the 758th base (A → T) as the detection site. ) Is preferably a partial sequence.

First, genomic DNA is isolated from whole blood. Isolation of genomic DNA from whole blood can be performed by a conventionally well-known method, For example, a commercial genomic DNA isolation kit (brand name GFX Genomic Blood DNA Purification kit; GE Healthcare Biosciences make) etc. can be used.

Next, the detection probe and the inhibition polynucleotide are added to the sample containing the isolated genomic DNA. The addition of the detection probe and the inhibition polynucleotide is not limited as described below. In this embodiment, as an example, a method of adding the inhibition polynucleotide after adding the detection probe is used as an example. have.

That is, a detection probe is first added to a sample containing isolated genomic DNA. As said detection probe, although the thing mentioned above is mentioned, for example, QProbe is especially preferable. As described above, this QProbe is a probe in which the terminal base is generally cytosine and the terminal is labeled with a fluorescent dye. By hybridizing to the sequence to be detected, the fluorescent dye interacts with guanine of the sequence to be detected, and as a result, fluorescence is reduced (or quenched).

As mentioned above, the sequence of the said detection probe should just be complementary to the detection object sequence containing a point mutation, and can be suitably designed according to the said detection object sequence. When detecting the point mutation (A-> T) of the 758th base A in the abl1 gene (SEQ ID NO: 1), the polynucleotide etc. which consist of the nucleotide sequence of sequence number 4 mentioned above are mentioned, for example.

Probe sequence number 4 for detection of variant A758T

5'-ccgaActggcccccgc-3 '(GC content 81.3%)

In the case where the gene amplification method is performed using DNA or RNA in the sample as a template, the timing of addition of the detection probe is not particularly limited. The detection probe may be added to, for example, the amplification product obtained after the gene amplification treatment described below, but is preferably added before the gene amplification treatment. When the detection probe is added before the gene amplification treatment as described above, for example, to prevent elongation of the probe itself, a phosphate group may be further added to the 3 'end thereof, and the 3' end is replaced with the fluorescent dye as described above. It may be labeled.

The detection probe may be added to, for example, a liquid sample containing isolated genomic DNA, or may be mixed with genomic DNA in a solvent. Examples of the solvent is not particularly limited, for example, those such as Tris-HCl buffer, KCl, MgCl _2, MgSO _4, glycerol, and the like, such as solvent, PCR reaction solution containing a conventionally known of.

Subsequently, a target sequence is amplified by gene amplification using the isolated genomic DNA as a template. Specifically, the sequence containing the base site | part which a point mutation for a detection object generate | occur | produces, ie, the sequence to be detected, and the sequence to which it is not detected are amplified.

The gene amplification method is not limited, and examples thereof include a polymerase chain reaction (PCR) method, a nucleic acid sequence based amplification (NASBA) method, a transcription-mediated amplification (TMA) method, and a strand displacement amplification (SDA) method. , PCR is preferred. In addition, although this invention is demonstrated below using the PCR method as an example, it does not restrict to this. In addition, the conditions of PCR are not specifically limited, It can carry out by a conventionally well-known method.

The sequence of the primer of PCR is not particularly limited as long as it can amplify a target detection target sequence, and can be appropriately designed by a conventionally known method according to the target sequence. The region to be amplified may be, for example, only a target detection target sequence or only a region including the detection target sequence. The length of the primer is not particularly limited and may be set to a general length, for example, 10 to 30 mer. As mentioned above, when detecting the point mutation (A → T) of the 758th base A in the abl1 gene (SEQ ID NO: 1), a primer consisting of the polynucleotide shown below can be used as a specific example. Combination of these primers is not particularly limited. When the polynucleotide (sense primer) consisting of the nucleotide sequence of SEQ ID NO: 9 and the polynucleotide (antisense primer) consisting of the nucleotide sequence of SEQ ID NO: 10 are combined, the length of the amplification product obtained is about 103 mer.

(Primer set 1)

Sense primer SEQ ID NO: 9

5'-ggagatggaacgcacggac-3 '(GC content 63.2%)

Antisense primer SEQ ID NO: 10

5'-ggccaccgtcaggctg-3 '(GC content 75%)

(Primer set 2)

Sense primer SEQ ID NO: 11

5'-gacaagtgggagatggaacgc-3 '

Antisense primer SEQ ID NO: 12

5'-cacggccaccgtcagg-3 '

Next, prior to the hybridization step described later, the inhibitory polynucleotide is added to the sample containing the amplification product. The addition ratio of the said polynucleotide for inhibition is as above-mentioned. As mentioned above, the timing of addition is not limited to this, For example, it can carry out before and behind or simultaneously with addition of the said detection probe. In addition, the addition of the inhibitory polynucleotide may be either before or after the gene amplification treatment described above, but for example, it is preferable to add the polynucleotide for inhibition before the gene amplification treatment.

As described above, when the inhibitory polynucleotide is added before the gene amplification treatment, for example, a phosphate group may be further added to the 3 'end to prevent the inhibition polynucleotide itself from elongating.

As described above, when detecting the point mutation (A → T) of the 758th base A in the abl1 gene (SEQ ID NO: 1), the inhibitory polynucleotide does not contain, for example, a point mutation, The polynucleotide which consists of a base sequence, etc. are mentioned. It is preferable to use this inhibitory polynucleotide in combination with the detection probe which consists of the nucleotide sequence of sequence number 4 mentioned above, for example.

Polynucleotide SEQ ID NO: 5 for inhibition of variant A758T

5'-cctccccg Tactggcccccg-3 '(GC content 80%)

Next, hybridization of the single-stranded DNA obtained by dissociation and dissociation of the obtained amplification product with the detection probe and the inhibition polynucleotide is carried out.

The heating temperature in the dissociation step is not particularly limited as long as it is a temperature at which the amplification product can dissociate. For example, the heating temperature is 85 ° C or higher, and preferably 85 ° C to 95 ° C. Heating time is not specifically limited, either, For example, they are 1 second-10 minutes, Preferably they are 1 second-5 minutes.

In addition, hybridization of the dissociated single-stranded DNA and the detection probe and hybridization of the single-stranded DNA and the inhibitory polynucleotide can be performed by, for example, lowering the heating temperature in the dissociation step after the dissociation step. Can be. As temperature conditions, it is 40 degrees C or less, for example.

The volume and concentration of each composition in the reaction solution of the hybridization step are not particularly limited. As a specific example, in the reaction solution, the DNA concentration is, for example, 0.01 to 1 µM, preferably 0.1 to 0.5 µM, and the concentration of the detection probe is, for example, in a range that satisfies the addition ratio to the DNA. For example, the concentration is 0.001 to 10 µM, preferably 0.001 to 1 µM, and the concentration of the inhibitory polynucleotide is, for example, 0.1 nM to 1 mM, preferably 0.1 nM to 100 µM.

In the present invention, as described above, addition of the inhibitory polynucleotide is important. For example, the concentration in the reaction solution of the DNA to be detected, the probe for detection, the inhibitory polynucleotide, etc. is not particularly limited. As a specific example, in the detection of a signal to be described later, for example, as the detection sensitivity of the device to be used is relatively high, the concentration of the DNA to be detected in the reaction solution can be reduced, and the detection sensitivity of the device to be used is relatively. It is preferable to increase the density | concentration of the DNA to be detected in reaction liquid, so that it is low. As a specific example, when a commercially available device (trade name Smart Cycler; manufactured by Cepheid) is used for signal detection described later, for example, in a reaction solution, a DNA concentration of 5 to 1000 nM and a probe concentration of 50 to 1000 nM are detected. The concentration of the polynucleotide for inhibition is preferably 5 nM to 100 μM, and more preferably, the DNA concentration for detection is 10 to 500 nM, the detection probe concentration for 100 to 500 nM, and the inhibition polynucleotide concentration for 10 nM. ˜50 μM.

Then, the formed hybridization of the single-stranded DNA and the labeled probe or the inhibitory polynucleotide is heated to measure the fluctuation of the signal due to the temperature rise. For example, when Q-Probe is used, fluorescence is reduced (or quenched) in the state of hybridization with single-stranded DNA, and fluoresces in the dissociated state. Therefore, for example, the hybrid formation in which fluorescence is reduced (or quenched) may be gradually heated to measure an increase in fluorescence intensity accompanying a temperature rise.

The temperature range when measuring signal variation is not particularly limited. The start temperature is, for example, room temperature (eg, 10 ° C.) to 85 ° C., preferably 25 to 70 ° C., and the end temperature is 40 to 105 ° C., for example. In addition, the rate of rise of temperature is not particularly limited, and is, for example, 0.1 to 20 ° C / sec, preferably 0.3 to 5 ° C / sec.

Next, the Tm value is determined by analyzing the fluctuation of the signal. Specifically, the value (-d fluorescence intensity increase amount / dt) at each temperature is calculated from the obtained fluorescence intensity, and the temperature showing the lowest value can be determined as the Tm value. Further, the point where the fluorescence intensity increase amount (fluorescence intensity increase amount / t) per unit time is the highest may be determined as the Tm value. In addition, when using the probe which does not show a signal by itself but shows a signal by hybrid formation instead of a quenching probe, the decrease amount of fluorescence intensity may be measured on the contrary.

The Tm value can be calculated, for example, by conventionally known MELTCALC software (http://www.meltcalc.com/) or the like, and can also be determined by the Nearest Neighbor Method.

In addition, in the present invention, as described above, instead of a method of measuring the signal fluctuation caused by heating the hybrid forming body by measuring the temperature rise, for example, the signal fluctuation at the time of hybrid formation may be measured. That is, when the temperature of the sample to which the probe is added is lowered to form a hybrid body, the signal variation caused by the temperature drop may be measured.

Specific examples are shown below. As a probe for detection, when a labeling probe (for example, Q-Probe) that exhibits a signal alone and does not exhibit a signal by hybrid formation is used, the probe dissociates when the detection probe is added to a sample. Although fluorescence is emitted, the fluorescence is reduced (or quenched) when a hybrid is formed according to a drop in temperature. Therefore, for example, the temperature of the sample may be gradually dropped to measure the decrease in fluorescence intensity caused by the temperature drop. On the other hand, when a labeling probe that does not display a signal alone and exhibits a signal by hybrid formation is used as the detection probe, when the probe is added to a sample, the probe dissociates and does not fluoresce, but When a hybrid is formed according to the drop, it fluoresces. Therefore, for example, the temperature of the sample may be gradually dropped to measure an increase in fluorescence intensity caused by the temperature drop.

Subsequently, the detection probe kit of the present invention, as described above, is a kit for use in the method for detecting the mutation of the present invention and is complementary to a detection probe consisting of a polynucleotide complementary to a detection target sequence and a non-detection target sequence. It characterized in that it comprises a polynucleotide for inhibition. In the present invention, the detection target sequence is a detection target DNA or a partial sequence thereof in which a detection site is mutated as described above, and is a sequence including the mutated detection site. As described above, the non-detection target sequence is the non-detection DNA or a partial sequence of which the detection site is not mutated and is a sequence including the undetected detection site. The detection probe kit of the present invention may include the probe and the inhibitory polynucleotide, and other configurations are not limited.

The probe and the inhibitory polynucleotide are not limited, and the same ones as described above can be used, and preferred combinations are also as described above.

In the detection probe kit of the present invention, the probe and the inhibitory polynucleotide may be mixed as one reagent or may be independent as separate reagents. In the former case, the probe and the inhibitory polynucleotide are preferably mixed in the same ratio as described above. In the latter case, for example, the ratio in the reaction solution may be used so as to be in the range described above. In addition, the detection probe kit of the present invention may further have a primer for amplifying a region including a sequence complementary to the probe.

Moreover, the data analysis method of this invention is a data analysis method for determining the presence or absence of the mutation of DNA, It is characterized by having the following (a)-(b) process.

(a) analyzing the signal variation obtained in step (C) in the variation detection method of the present invention and calculating the Tm value,

(b) A step of determining the presence or absence of DNA variation from the Tm value calculated in the calculation step.

The system of the present invention is a system for detecting the presence or absence of variation in DNA in a sample, the input means for inputting the signal variation obtained in step (C) in the method for detecting variation in the sample, the input means according to the input means And a determining means for determining the presence or absence of the variation of DNA based on the calculating means for calculating the Tm value from the signal variation and the Tm value calculated by the calculating means. Examples of the system of the present invention include a detection device constructed by a computer system. The hardware structure of the system is not limited, and for example, an input device such as a storage device, a keyboard or a mouse is connected to a CPU serving as a control unit, and for example, a display device for displaying a result output device, input data or a result. (Display) or the like may be connected. In addition, each means should just be a functional block implemented, for example by the CPU of a computer executing a predetermined program. For this reason, for example, each configuration means may not be actually attached as hardware, or may be a network system.

Moreover, the system of this invention may have a means for performing the process (B) and (C) in the detection method of the mutation of this invention, for example. As a means for performing the said process, a temperature control means and a signal detection means are mentioned, for example. By the execution of the temperature control means, for example, formation of double-stranded DNA (hybrid forming body) by hybridization and dissociation of the hybrid forming body can be performed. Further, by the signal detecting means, for example, the amount of signal fluctuation due to the formation of the hybrid formation in response to temperature changes or the amount of signal fluctuation due to the dissociation of the hybrid formation can be detected. In this case, for example, instead of the input means, it may have recording means for recording the signal variation detected by the signal detecting means. The system of the present invention may further comprise means for outputting the presence or absence of the mutation of the DNA determined by the determination means.

The program of the present invention is a computer program that can execute the data interpretation method of the present invention on a computer.

The electronic medium of the present invention is a computer-readable electronic medium (also referred to as a "recording medium") in which the computer program of the present invention is stored.

Next, the Example of this invention is described with a comparative example. However, the present invention is not limited by the following examples and comparative examples.

Example 1

758th point mutation in the abl1 gene (A → T)

(1) The polynucleotide for inhibition was added, and the Tm analysis of the point mutation (A-> T) of the 758th base in the abl1 gene was performed.

genomic DNA of the leukocyte cell line having no mutation at the 758th base of the abl1 gene (SEQ ID NO: 1) and genomic DNA of the leukocyte cell line having the mutation at the 758th base of the abl1 gene (the 758th base in SEQ ID NO: 1 are T: abl1 tyrosine kinase A758T (= Y253F)] was prepared. Hereinafter, the former which does not have a mutation is called "wtDNA", and the latter which has a mutation is called "mtDNA". And both were prepared at a predetermined ratio (mtDNA: wtDNA = 100: 0, 50:50, 10:90, 5:95, 3:97, 0: 100) to prepare 10 ⁴ copy / test (1 μl). PCR reaction was carried out by adding to 50 µl of the following PCR reaction solution. The final concentration of the inhibitory polynucleotide in the PCR reaction solution was 200 nM and the final concentration of the detection probe was 50 nM. The PCR reaction was performed by a thermal cycler for 60 seconds at 95 ° C., followed by 50 cycles of 95 ° C. 10 seconds and 60 ° C. 30 seconds as one cycle, followed by treatment at 95 ° C. for 1 second and 40 ° C. for 60 seconds. did. Subsequently, the PCR reaction solution was heated from 40 ° C. to 95 ° C. at a rate of temperature rise of 1 ° C./3 sec., And the change in fluorescence intensity over time was measured. The measurement wavelength was 585-700 nm. In addition, the addition ratio of the detection probe is 0.1 times in molar ratio with respect to the PCR amplification product, and the addition ratio of the inhibition polynucleotide is 0.4 times in molar ratio with respect to the PCR amplification product and 4 times in molar ratio with respect to the probe. In addition, the addition ratio with respect to the detection target DNA (amplification product of a detection target sequence) of a detection probe is as follows. These results are referred to as Example 1-1. In Comparative Example 1-1, fluorescence intensity was measured in the same manner except that 2 μl of distilled water was added to the PCR reaction solution instead of 2 μl of the inhibition polynucleotide.

[Table 1]

Sample
(mtDNA: wtDNA) Addition ratio to DNA to be detected 100: 0
50:50
10:90
5:95
3:97
0: 100 0.1x
0.2x
1x
Twice
3.3x
-

TABLE 2

(PCR reaction solution: unit μl) Distilled water
10 × gene Taq buffer *
40% Glycerol
2.5 mM dNTP
100 uM sense primer
100 uM antisense primer
5 uM detection probe
5 uM Inhibition Polynucleotide
5 U / μl Gene Taq FP * 34.375
5
3.125
4
0.5
0.25
0.5
2
0.25 Total amount 50 μl

* Trademark Gene Taq FP: manufactured by Nippon Jinjin Co., Ltd.

Sense primer SEQ ID NO: 9

5'-ggagatggaacgcacggac-3 '

Antisense primer SEQ ID NO: 10

5'-ggccaccgtcaggctg-3 '

Detection probe sequence number 4

5 '-(TAMRA) -ccgAactggcccccgc-P-3'

Polynucleotide SEQ ID NO: 5 for Inhibition

5'-cctccccgTactggcccccg-3 '

These results are shown in FIG. 1 and FIG. Both figures are Tm analysis graph which shows the change of fluorescence intensity with temperature rise, and the derivative value of a vertical axis shows "-d fluorescence intensity increase amount / dt" (it is the same hereafter). 1 is a result of Example 1-1 and FIG. 2 of Comparative Example 1-1. In Figure 1, (A) 100% of the whole genome has a point mutation, (B) 5% of the whole genome has a point mutation, (C) 3% of the whole genome has a point mutation The sample with (D) is the result for the sample where the whole genome does not have a point mutation. In Figure 2, (A) 100% of the whole genome has a point mutation, (B) 50% of the whole genome has a point mutation, (C) 5% of the whole genome has a point mutation The sample, (D), is a result for a sample in which 3% of the whole genome has a point mutation, and (E) is for a sample in which the whole genome does not have a point mutation.

As shown in Fig. 1 (A) and Fig. 2 (A), the peak of mtDNA was detected at 69.5 ° C, and as shown in Fig. 1 (D) and Fig. 2E, the peak peak of the signal was detected at 61.5 ° C. . Each sample was evaluated based on this. As a result, in Comparative Example 1-1 in which the inhibitory polynucleotide was not added, when the mtDNA was 50%, the signal of mtDNA can be detected as shown in FIG. , 3%), the signal of mtDNA could not be detected at all as shown to FIG. 2 (C) and (D). On the other hand, in Example 1-1 to which the inhibitory polynucleotide was added, even if the amount of mtDNA was small (5%, 3%), as shown in Fig. 1 (B) and (C), the signal of mtDNA could be detected. Could. That is, the addition of the inhibitory polynucleotide inhibited the hybridization of the detection probe to the wtDNA without the point mutation. As a result, the amount of the detection probe that binds to mtDNA is increased, thereby improving the detection sensitivity. I can say that.

(2) In addition, the addition ratio of the detection probe was changed, and Tm analysis was performed for the point mutation (A → T) of the 758th base in the abl1 gene.

The final concentration of the detection probe was set to 10 nM of 1/5 except that 0.5 μl of the probe addition amount for detection of the PCR reaction solution in Example 1 was set to 0.1 μl and 34.375 μl of the distilled water addition amount was 34.775 μl. In the same manner as in Example 1, Tm analysis was performed. This result is shown in FIG. 3 as Example 1-2. In FIG. 3, (A) is a sample in which 100% of the whole genome has a point mutation, (B) is a sample in which 10% of the whole genome has a point mutation, and (C) is 5% of the whole genome has a point mutation. Sample, (D), is the result for samples where 3% of the total genome had a point mutation.

As a result, as shown in the above figure, the peak of mtDNA was remarkably detected. For example, when compared with the result of Example 1-1 which performed the analysis on the same conditions except the addition amount of a probe, it shows in FIG.3 (C) and the above-mentioned FIG.1 (B), FIG.3 (D), and FIG.1 (C). As described above, by reducing the amount of probe added compared to Example 1-1 (0.02 times in molar ratio with respect to PCR amplification products), the peak of wtDNA was further reduced, and the relative peak of mtDNA was increased. In this respect, it was found that the detection sensitivity was further improved by adjusting the amount of the probe added.

(3) The addition amount of the inhibitory polynucleotide was increased, and the Tm analysis was performed for the point mutation (A → T) of the 758th base in the abl1 gene.

The Tm analysis was carried out in the same manner as in Example 1-2 of (2), except that 3 µl of the polynucleotide addition amount for inhibition of the PCR reaction solution in Example 1 and 33.775 µl of the amount of 34.375 µl of distilled water were added. Done. The final concentration of the inhibitory polynucleotide in the PCR reaction solution is 300 nM, and the final concentration of the detection probe is 10 nM. These results are shown in FIG. 4 as Example 1-3. In Figure 4, (A) 100% of the whole genome has a point mutation, (B) 10% of the whole genome has a point mutation, (C) 5% of the whole genome has a point mutation Sample, (D), is the result for samples where 3% of the total genome had a point mutation.

As a result, as shown in FIG. 4, the peak of mtDNA was detected very remarkably. In particular, compared with the result of Example 1-2 which analyzed on the same conditions except the addition amount of the polynucleotide for inhibition, FIG. 4 (B), FIG. 3 (B), FIG. 4 (C), and FIG. 3 (C) 4, (D) and 3 (D), the addition amount of the inhibitory polynucleotide was increased by more than Example 1-2 (30-fold in molar ratio with respect to the probe), and the peak of wtDNA was further increased. Decreases, and the relative peak of mtDNA is increasing. From this point of view, it was found that the detection sensitivity is further improved by adjusting the amount of the polynucleotide for inhibition to be added.

[Example 2]

Point mutation of the 756th base of the abl1 gene (G → C)

The polynucleotide for inhibition was added, and the Tm analysis of the point mutation (G → C) of the 756th base in the abl1 gene was performed.

A plasmid into which the normal abl1 gene sequence having no mutation is inserted in the 756th base G shown in SEQ ID NO: 1, and the mutant abl1 gene (abl1 tyrosine kinase G756C (= Q250E)) in which the 756th base G has been changed to C. The inserted plasmid was prepared. Hereinafter, the former which does not have a mutation is called "wtDNA", and the latter which has a mutation is called "mtDNA". In the same manner as in Table 1 of Example 1, both were prepared at a predetermined ratio (mtDNA: wtDNA = 0: 100, 3:97, 100: 0) to give 10 ⁴ copy / test (1 μl). It was added to 49 microliters of PCR reaction liquids, and PCR reaction was performed. In the PCR reaction solution, the final concentration of the inhibitory polynucleotide was 300 nM and the final concentration of the detection probe was 50 nM. The PCR reaction was performed by a thermal cycler for 60 seconds at 95 ° C., followed by 50 cycles of 95 ° C. 1 second and 58 ° C. 30 seconds as one cycle, and at 95 ° C. for 1 second and 40 ° C. for 60 seconds. Processed. Subsequently, the PCR reaction solution was heated from 40 ° C. to 95 ° C. with a rate of temperature rise of 1 ° C./3 sec., And the change in fluorescence intensity over time was measured. The measurement wavelength was 585-700 nm. The addition ratio of the detection probe is 0.05 times in molar ratio with respect to the PCR amplification product, and the addition ratio of the inhibition polynucleotide is 0.3 times in molar ratio with respect to the PCR amplification product and 6 times in molar ratio with respect to the probe. As Comparative Example 2, fluorescence intensity was measured in the same manner as in the following PCR reaction solution, except that 3 μl of distilled water was added instead of 3 μl of the inhibiting polynucleotide.

[Table 3]

(PCR reaction solution: unit μl) Distilled water
10 × gene Taq buffer *
40% Glycerol
2.5 mM dNTP
100 mM MgCl ₂
100 uM sense primer
100 uM antisense primer
5 uM detection probe
5 uM Inhibition Polynucleotide
5 U / μl Gene Taq FP * 21.75
5
12.5
4
0.5
One
0.5
0.5
3
0.25 Total amount 49 μl

Sense primer SEQ ID NO: 13

5'-gacaagtgggagatggaacgc-3 '

Antisense primer SEQ ID NO: 14

5'-cacggccaccgtcagg-3 '

Detection probe sequence number 2

5 '-(BODIPYFL) -ccgtaGtggcccccgc-P-3'

Polynucleotide SEQ ID NO: 3 for Inhibition

5'-ccgtaCtggcccccgc-P-3 '

These results are shown in FIG. 5 is a graph of the Tm analysis showing the change in fluorescence intensity with increasing temperature. In FIG. 5, (A) is a sample in which the entire plasmid does not have a point mutation, (B) is a sample in which 100% of the whole plasmid has a point mutation, and (C) and (D) are 3% in a total plasmid. Samples with mutations. And (C) is the result of Comparative Example 2, (D) is the result of Example 2.

As shown in Fig. 5A, the wtDNA was 61.0 ° C, and as shown in Fig. 5B, the mtDNA was 67.0 ° C, and peaks of the signals were detected. Using this as a standard, samples with 3% of all plasmids having point mutations were evaluated. As a result, as shown in Fig. 5C, in Comparative Example 2 in which no inhibitory polynucleotide was added, no signal of mtDNA could be detected. On the other hand, in Example 2 to which the inhibitory polynucleotide was added, as shown in FIG. 5 (D), even if the amount of mtDNA was small, signals of both wtDNA and mtDNA could be detected.

[Example 3]

Point mutation at base 763 of abl1 gene (G → A)

The polynucleotide for inhibition was added, and the Tm analysis of the point mutation (G-> A) of the 763th base in the abl1 gene was performed.

A plasmid in which the normal abl1 gene sequence having no mutation is inserted in the 763th base G shown in SEQ ID NO: 1, and the mutant abl1 gene (abl1 tyrosine kinase G763A (= E255K)) in which the 763th base G is changed to A. The inserted plasmid was prepared. Hereinafter, the former which does not have a mutation is called "wtDNA", and the latter which has a mutation is called "mtDNA". In the same manner as in Table 1 of Example 1, both were prepared at a predetermined ratio (mtDNA: wtDNA = 0: 100, 3:97, 100: 0) to give 10 ⁴ copy / test (1 μl). It was added to 49 microliters of PCR reaction liquids, and PCR reaction was performed. In the PCR reaction solution, the final concentration of the inhibitory polynucleotide was 500 nM and the final concentration of the detection probe was 50 nM. The PCR reaction was carried out in the same manner as in Example 2, and the change in fluorescence intensity over time was measured. The measurement wavelength was 585-700 nm. The addition ratio of the detection probe is 0.05 times in molar ratio with respect to the PCR amplification product, and the addition ratio of the inhibition polynucleotide is 0.5 times in molar ratio with respect to the PCR amplification product and 10 times in molar ratio with respect to the probe. As Comparative Example 3, fluorescence intensity was measured in the same manner as in the following PCR reaction solution, except that 5 µl of distilled water was added instead of 5 µl of the inhibiting polynucleotide.

[Table 4]

(PCR reaction solution: unit μl) Distilled water
10 × gene Taq buffer *
40% Glycerol
2.5 mM dNTP
100 uM sense primer
100 uM antisense primer
5 uM detection probe
5 uM Inhibition Polynucleotide
5 U / μl Gene Taq FP * 23.375
5
9.375
4
0.5
One
0.5
5
0.25 Total amount 49 μl

Sense primer SEQ ID NO: 15

5'-gacaagtgggagatggaacgc-3 '

Antisense primer SEQ ID NO: 16

5'-cacggccaccgtcagg-3 '

Detection probe SEQ ID NO: 7

5 '-(BODIPY FL) -ccagtacgggAaggtgt-P-3'

Inhibition Polynucleotide SEQ ID NO: 8

5'-ccagtacgggGaggtgt-P-3 '

These results are shown in FIG. 6 is a graph of Tm analysis showing the change in fluorescence intensity with increasing temperature. In Figure 6, (A) is a sample in which the entire plasmid does not have a point mutation, (B) is a sample in which 100% of the whole plasmid has a point mutation, and (C) and (D) are 3% in a total plasmid. Samples with mutations. And (C) is the result of Comparative Example 3, (D) is the result of Example 3.

As shown in Fig. 6A, the wtDNA was 50.0 ° C, and as shown in Fig. 6B, the mtDNA was 59.0 ° C, and peaks of the signals were detected. Using this as a standard, samples with 3% of all plasmids having point mutations were evaluated. As a result, as shown in Fig. 6C, in Comparative Example 3 in which no inhibitory polynucleotide was added, no signal of mtDNA could be detected. In contrast, in Example 3 to which the polynucleotide for inhibition was added, as shown in FIG. 6 (D), even if a small amount of mtDNA was detected, signals of both wtDNA and mtDNA could be detected.

Example 4

Point mutation of the 758th base of the abl1 gene (A → T)

The polynucleotide for inhibition was added and the Tm analysis was performed for the point mutation (A → T) of the 758th base in the abl1 gene.

A plasmid in which the normal abl1 gene sequence having no mutation is inserted in the 758th base A shown in SEQ ID NO: 1, and a mutant abl1 gene (abl1 tyrosine kinase A758T (= Y253F)) in which the 758th base A has been changed to T. The inserted plasmid was prepared. Hereinafter, the former which does not have a mutation is called "wtDNA", and the latter which has a mutation is called "mtDNA". In the same manner as in Table 1 of Example 1, both were prepared at a predetermined ratio (mtDNA: wtDNA = 0: 100, 3:97, 100: 0) to give 10 ⁴ copy / test (1 μl). It was added to 49 microliters of PCR reaction liquids, and PCR reaction was performed. In the PCR reaction solution, the final concentration of the inhibitory polynucleotide was 500 nM and the final concentration of the detection probe was 50 nM. The PCR reaction was carried out in the same manner as in Example 2, and the change in fluorescence intensity over time was measured. The measurement wavelength was 585-700 nm. The addition ratio of the detection probe is 0.05 times in molar ratio with respect to the PCR amplification product, and the addition ratio of the inhibition polynucleotide is 0.5 times in molar ratio with respect to the PCR amplification product and 10 times in molar ratio with respect to the probe. In Comparative Example 4, the fluorescence intensity was measured in the same manner except that 5 μl of distilled water was added to the following PCR reaction solution instead of 5 μl of the inhibiting polynucleotide.

[Table 5]

(PCR reaction solution: unit μl) Distilled water
10 × gene Taq buffer *
40% Glycerol
2.5 mM dNTP
100 uM sense primer
100 uM antisense primer
5 uM detection probe
5 uM Inhibition Polynucleotide
5 U / μl Gene Taq FP * 20.25
5
12.5
4
One
0.5
0.5
5
0.25 Total amount 49 μl

Sense primer SEQ ID NO: 11

5'-gacaagtgggagatggaacgc-3 '

Antisense primer SEQ ID NO: 12

5'-cacggccaccgtcagg-3 '

Detection probe sequence number 4

5 '-(TAMRA) -ccgAactggcccccgc-P-3'

Polynucleotide SEQ ID NO: 6 for Inhibition

5'-ccg Tactggcccccgc-P-3 '

These results are shown in FIG. 7 is a graph of Tm analysis showing the change in fluorescence intensity with temperature rise. In FIG. 7, (A) is a sample in which the entire plasmid does not have a point mutation, (B) is a sample in which 100% of the whole plasmid has a point mutation, and (C) and (D) are 3% in a total plasmid. Samples with mutations. And (C) is the result of Comparative Example 4, (D) is the result of Example 4.

As shown in Fig. 7A, the wtDNA was 60.0 ° C, and as shown in Fig. 7B, the mtDNA was 67.0 ° C, and peaks of the signals were detected. Using this as a standard, 3% of the plasmids were sampled with point mutations. As shown in Fig. 7C, in Comparative Example 4 in which no inhibitory polynucleotide was added, no signal of mtDNA was detected. I could not. On the other hand, in Example 4 to which the inhibitory polynucleotide was added, as shown in FIG. 7 (D), even if the amount of mtDNA was small, signals of both wtDNA and mtDNA could be detected.

As described above, according to the mutation detection method of the present invention, by adding a polynucleotide for inhibition as described above, for example, the target DNA having a point mutation and the ratio without the point mutation, such as a leukocyte sample of a leukemia patient Even if the DNA to be detected is mixed, the point mutation can be detected with excellent sensitivity. Moreover, since the kind of mutation can be specified, it is a detection method excellent also in specificity. Therefore, this method is particularly useful for the detection of point mutations in leukemia patients. For example, the method can be interpreted at the genetic level as to whether or not leukemia therapeutic drugs are suitable for an individual, and thus it is a very useful method in the medical field. have.

                         SEQUENCE LISTING <110> ARKRAY, Inc. <120> Method for detection of mutation and kit therefor <130> TF07027-01 <150> JP2006 / 216194 <151> 2006-08-08 <150> JP2007 / 040078 <151> 2007-02-20 <160> 16 <170> PatentIn version 3.1 <210> 1 <211> 5381 <212> DNA <213> Homo sapiens <400> 1 atgttggaga tctgcctgaa gctggtgggc tgcaaatcca agaaggggct gtcctcgtcc 60 tccagctgtt atctggaaga agcccttcag cggccagtag catctgactt tgagcctcag 120 ggtctgagtg aagccgctcg ttggaactcc aaggaaaacc ttctcgctgg acccagtgaa 180 aatgacccca accttttcgt tgcactgtat gattttgtgg ccagtggaga taacactcta 240 agcataacta aaggtgaaaa gctccgggtc ttaggctata atcacaatgg ggaatggtgt 300 gaagcccaaa ccaaaaatgg ccaaggctgg gtcccaagca actacatcac gccagtcaac 360 agtctggaga aacactcctg gtaccatggg cctgtgtccc gcaatgccgc tgagtatctg 420 ctgagcagcg ggatcaatgg cagcttcttg gtgcgtgaga gtgagagcag tcctggccag 480 aggtccatct cgctgagata cgaagggagg gtgtaccatt acaggatcaa cactgcttct 540 gatggcaagc tctacgtctc ctccgagagc cgcttcaaca ccctggccga gttggttcat 600 catcattcaa cggtggccga cgggctcatc accacgctcc attatccagc cccaaagcgc 660 aacaagccca ctgtctatgg tgtgtccccc aactacgaca agtgggagat ggaacgcacg 720 gacatcacca tgaagcacaa gctgggcggg ggccagtacg gggaggtgta cgagggcgtg 780 tggaagaaat acagcctgac ggtggccgtg aagaccttga aggaggacac catggaggtg 840 gaagagttct tgaaagaagc tgcagtcatg aaagagatca aacaccctaa cctggtgcag 900 ctccttgggg tctgcacccg ggagcccccg ttctatatca tcactgagtt catgacctac 960 gggaacctcc tggactacct gagggagtgc aaccggcagg aggtgaacgc cgtggtgctg 1020 ctgtacatgg ccactcagat ctcgtcagcc atggagtacc tggagaagaa aaacttcatc 1080 cacagagatc ttgctgcccg aaactgcctg gtaggggaga accacttggt gaaggtagct 1140 gattttggcc tgagcaggtt gatgacaggg gacacctaca cagcccatgc tggagccaag 1200 ttccccatca aatggactgc acccgagagc ctggcctaca acaagttctc catcaagtcc 1260 gacgtctggg catttggagt attgctttgg gaaattgcta cctatggcat gtccccttac 1320 ccgggaattg acctgtccca ggtgtatgag ctgctagaga aggactaccg catggagcgc 1380 ccagaaggct gcccagagaa ggtctatgaa ctcatgcgag catgttggca gtggaatccc 1440 tctgaccggc cctcctttgc tgaaatccac caagcctttg aaacaatgtt ccaggaatcc 1500 agtatctcag acgaagtgga aaaggagctg gggaaacaag gcgtccgtgg ggctgtgagt 1560 accttgctgc aggccccaga gctgcccacc aagacgagga cctccaggag agctgcagag 1620 cacagagaca ccactgacgt gcctgagatg cctcactcca agggccaggg agagagcgat 1680 cctctggacc atgagcctgc cgtgtctcca ttgctccctc gaaaagagcg aggtcccccg 1740 gagggcggcc tgaatgaaga tgagcgcctt ctccccaaag acaaaaagac caacttgttc 1800 agcgccttga tcaagaagaa gaagaagaca gccccaaccc ctcccaaacg cagcagctcc 1860 ttccgggaga tggacggcca gccggagcgc agaggggccg gcgaggaaga gggccgagac 1920 atcagcaacg gggcactggc tttcaccccc ttggacacag ctgacccagc caagtcccca 1980 aagcccagca atggggctgg ggtccccaat ggagccctcc gggagtccgg gggctcaggc 2040 ttccggtctc cccacctgtg gaagaagtcc agcacgctga ccagcagccg cctagccacc 2100 ggcgaggagg agggcggtgg cagctccagc aagcgcttcc tgcgctcttg ctccgcctcc 2160 tgcgttcccc atggggccaa ggacacggag tggaggtcag tcacgctgcc tcgggacttg 2220 cagtccacgg gaagacagtt tgactcgtcc acatttggag ggcacaaaag tgagaagccg 2280 gctctgcctc ggaagagggc aggggagaac aggtctgacc aggtgacccg aggcacagta 2340 acgcctcccc ccaggctggt gaaaaagaat gaggaagctg ctgatgaggt cttcaaagac 2400 atcatggagt ccagcccggg ctccagcccg cccaacctga ctccaaaacc cctccggcgg 2460 caggtcaccg tggcccctgc ctcgggcctc ccccacaagg aagaagctgg aaagggcagt 2520 gccttaggga cccctgctgc agctgagcca gtgaccccca ccagcaaagc aggctcaggt 2580 gcaccagggg gcaccagcaa gggccccgcc gaggagtcca gagtgaggag gcacaagcac 2640 tcctctgagt cgccagggag ggacaagggg aaattgtcca ggctcaaacc tgccccgccg 2700 cccccaccag cagcctctgc agggaaggct ggaggaaagc cctcgcagag cccgagccag 2760 gaggcggccg gggaggcagt cctgggcgca aagacaaaag ccacgagtct ggttgatgct 2820 gtgaacagtg acgctgccaa gcccagccag ccgggagagg gcctcaaaaa gcccgtgctc 2880 ccggccactc caaagccaca gtccgccaag ccgtcgggga cccccatcag cccagccccc 2940 gttccctcca cgttgccatc agcatcctcg gccctggcag gggaccagcc gtcttccacc 3000 gccttcatcc ctctcatatc aacccgagtg tctcttcgga aaacccgcca gcctccagag 3060 cggatcgcca gcggcgccat caccaagggc gtggtcctgg acagcaccga ggcgctgtgc 3120 ctcgccatct ctaggaactc cgagcagatg gccagccaca gcgcagtgct ggaggccggc 3180 aaaaacctct acacgttctg cgtgagctat gtggattcca tccagcaaat gaggaacaag 3240 tttgccttcc gagaggccat caacaaactg gagaataatc tccgggagct tcagatctgc 3300 ccggcgacag caggcagtgg tccagcggcc actcaggact tcagcaagct cctcagttcg 3360 gtgaaggaaa tcagtgacat agtgcagagg tagcagcagt caggggtcag gtgtcaggcc 3420 cgtcggagct gcctgcagca catgcgggct cgcccatacc cgtgacagtg gctgacaagg 3480 gactagtgag tcagcacctt ggcccaggag ctctgcgcca ggcagagctg agggccctgt 3540 ggagtccagc tctactacct acgtttgcac cgcctgccct cccgcacctt cctcctcccc 3600 gctccgtctc tgtcctcgaa ttttatctgt ggagttcctg ctccgtggac tgcagtcggc 3660 atgccaggac ccgccagccc cgctcccacc tagtgcccca gactgagctc tccaggccag 3720 gtgggaacgg ctgatgtgga ctgtcttttt catttttttc tctctggagc ccctcctccc 3780 ccggctgggc ctccttcttc cacttctcca agaatggaag cctgaactga ggccttgtgt 3840 gtcaggccct ctgcctgcac tccctggcct tgcccgtcgt gtgctgaaga catgtttcaa 3900 gaaccgcatt tcgggaaggg catgcacggg catgcacacg gctggtcact ctgccctctg 3960 ctgctgcccg gggtggggtg cactcgccat ttcctcacgt gcaggacagc tcttgatttg 4020 ggtggaaaac agggtgctaa agccaaccag cctttgggtc ctgggcaggt gggagctgaa 4080 aaggatcgag gcatggggca tgtcctttcc atctgtccac atccccagag cccagctctt 4140 gctctcttgt gacgtgcact gtgaatcctg gcaagaaagc ttgagtctca agggtggcag 4200 gtcactgtca ctgccgacat ccctccccca gcagaatgga ggcaggggac aagggaggca 4260 gtggctagtg gggtgaacag ctggtgccaa atagccccag actgggccca ggcaggtctg 4320 caagggccca gagtgaaccg tcctttcaca catctgggtg ccctgaaagg gcccttcccc 4380 tcccccactc ctctaagaca aagtagattc ttacaaggcc ctttcctttg gaacaagaca 4440 gccttcactt ttctgagttc ttgaagcatt tcaaagccct gcctctgtgt agccgccctg 4500 agagagaata gagctgccac tgggcacctg cgcacaggtg ggaggaaagg gcctggccag 4560 tcctggtcct ggctgcactc ttgaactggg cgaatgtctt atttaattac cgtgagtgac 4620 atagcctcat gttctgtggg ggtcatcagg gagggttagg aaaaccacaa acggagcccc 4680 tgaaagcctc acgtatttca cagagcacgc ctgccatctt ctccccgagg ctgccccagg 4740 ccggagccca gatacggggg ctgtgactct gggcagggac ccggggtctc ctggaccttg 4800 acagagcagc taactccgag agcagtgggc aggtggccgc ccctgaggct tcacgccggg 4860 agaagccacc ttcccacccc ttcataccgc ctcgtgccag cagcctcgca caggccctag 4920 ctttacgctc atcacctaaa cttgtacttt atttttctga tagaaatggt ttcctctgga 4980 tcgttttatg cggttcttac agcacatcac ctctttgccc ccgacggctg tgacgcagcc 5040 ggagggaggc actagtcacc gacagcggcc ttgaagacag agcaaagcgc ccacccaggt 5100 cccccgactg cctgtctcca tgaggtactg gtcccttcct tttgttaacg tgatgtgcca 5160 ctatatttta cacgtatctc ttggtatgca tcttttatag acgctctttt ctaagtggcg 5220 tgtgcatagc gtcctgccct gccccctcgg gggcctgtgg tggctccccc tctgcttctc 5280 ggggtccagt gcattttgtt tctgtatatg attctctgtg gttttttttg aatccaaatc 5340 tgtcctctgt agtatttttt aaataaatca gtgtttacat t 5381 <210> 2 <211> 16 <212> DNA <213> Artificial <220> <223> probe <400> 2 ccgtagtggc ccccgc 16 <210> 3 <211> 16 <212> DNA <213> Artificial <220> <223> polynucleotide <400> 3 ccgtactggc ccccgc 16 <210> 4 <211> 16 <212> DNA <213> Artificial <220> <223> probe <400> 4 ccgaactggc ccccgc 16 <210> 5 <211> 20 <212> DNA <213> Artificial <220> <223> polynucleotide <400> 5 cctccccgta ctggcccccg 20 <210> 6 <211> 16 <212> DNA <213> Artificial <220> <223> polynucleotide <400> 6 ccgtactggc ccccgc 16 <210> 7 <211> 17 <212> DNA <213> Artificial <220> <223> purobe <400> 7 ccagtacggg aaggtgt 17 <210> 8 <211> 17 <212> DNA <213> Artificial <220> <223> polynucleotide <400> 8 ccagtacggg gaggtgt 17 <210> 9 <211> 19 <212> DNA <213> Artificial <220> <223> sense primer <400> 9 ggagatggaa cgcacggac 19 <210> 10 <211> 16 <212> DNA <213> Artificial <220> <223> antisense primer <400> 10 ggccaccgtc aggctg 16 <210> 11 <211> 21 <212> DNA <213> Artificial <220> <223> sense primer <400> 11 gacaagtggg agatggaacg c 21 <210> 12 <211> 16 <212> DNA <213> Artificial <220> <223> antisense primer <400> 12 cacggccacc gtcagg 16 <210> 13 <211> 21 <212> DNA <213> Artificial <220> <223> sense primer <400> 13 gacaagtggg agatggaacg c 21 <210> 14 <211> 16 <212> DNA <213> Artificial <220> <223> antisense primer <400> 14 cacggccacc gtcagg 16 <210> 15 <211> 21 <212> DNA <213> Artificial <220> <223> sense primer <400> 15 gacaagtggg agatggaacg c 21 <210> 16 <211> 16 <212> DNA <213> Artificial <220> <223> antisense primer <400> 16 cacggccacc gtcagg 16  

Claims (34)

As a method of detecting a variation of DNA in a sample, The sample is a sample containing the DNA to be detected in which the detection site is mutated and the non-detection DNA in which the detection site is not mutated, A method for detecting variation comprising the following steps (A) to (E): (A) A step of adding a polynucleotide for inhibition that inhibits hybridization of a detection probe consisting of a polynucleotide complementary to a detection target sequence and a detection probe to a non-detection sequence to a sample containing the DNA, The detection target sequence includes the mutated detection site as the detection target DNA or a partial sequence thereof, The non-detection sequence is the non-detection DNA or a partial sequence thereof, and includes the undetected detection site, (B) hybridizing the detection probe to the DNA; (C) measuring the fluctuation of the signal according to the temperature change for the hybrid formation of the DNA and the detection probe, (D) analyzing the fluctuation of the signal to determine the Tm value, (E) Process of determining presence or absence of the mutation in the said detection object site | part from the said Tm value. The method of claim 1, wherein the detection probe is a probe labeled with a labeling substance. The method according to claim 2, wherein the labeling probe is a labeling probe that alone shows a signal and does not show a signal by hybrid formation, or a labeling probe that does not show a signal alone and also shows a signal by hybrid formation. The method of claim 2, wherein the labeling substance is a fluorescent dye and the labeling probe is a fluorescence alone and a fluorescence decreases by hybrid formation. The method according to claim 2, wherein the labeling probe is cytosine at its 3 'end, and the 3' end is a labeled probe. The method of claim 2, wherein the 5′-terminal base is cytosine and the 5′-terminal is labeled probe. The inhibitory polynucleotide of claim 1, wherein the inhibitory polynucleotide that inhibits hybridization of the detection probe complementary to the detection target sequence and the detection probe to the non-detection target sequence is identical except for a site paired with the detection site. Detection method of phosphorus variation. The method of claim 1, wherein the addition ratio of the inhibitory polynucleotide that inhibits hybridization of the detection probe to the non-detection target sequence is 0.1 to 100-fold in molar ratio relative to the detection probe. The method of claim 1, wherein the addition ratio of the detection probe is one or less times the molar ratio with respect to the DNA. The method of claim 1, wherein the DNA is DNA derived from white blood cells. The method of claim 1, wherein the mutation is a mutation of the abl1 gene. The method of claim 1, wherein the mutation is a change from the base sequence of SEQ ID NO: 1 to the C of the 756 th base G. 3. The method according to claim 12, wherein the detection probe is a probe consisting of the nucleotide sequence of SEQ ID NO: 2, the inhibitory polynucleotide to inhibit the hybridization of the detection probe to the non-detection target sequence is a nucleotide sequence of SEQ ID NO: A method for detecting a mutation which is a polynucleotide. The method of claim 1, wherein the mutation is a nucleotide of SEQ ID NO: 1 from the 758th base A to T. 7. The method according to claim 14, wherein the detection probe is a probe consisting of the nucleotide sequence of SEQ ID NO: 4, the inhibitory polynucleotide to inhibit the hybridization of the detection probe to the non-detection target sequence is SEQ ID NO: 5 and SEQ ID NO: 6 A method for detecting a mutation, wherein the mutation is a polynucleotide consisting of at least one nucleotide sequence. The method of claim 1, wherein the mutation is a mutation from base sequence 1 of SEQ ID NO: 1 to base G63 of A. 7. The method according to claim 16, wherein the detection probe is a probe consisting of the nucleotide sequence of SEQ ID NO: 7, and the inhibitory polynucleotide to inhibit the hybridization of the detection probe to the non-detection target sequence is a nucleotide sequence of SEQ ID NO: A method for detecting a mutation which is a polynucleotide. The method of claim 1, wherein the DNA in the sample is double-stranded DNA, and the step of dissociating the double-stranded DNA in the sample by heating prior to the step (B). The method for detecting mutation according to claim 1, wherein the DNA is an amplification product amplified by a gene amplification method using DNA from the sample as a template. The mutation detection method according to claim 1, wherein the DNA is an amplification product amplified by gene amplification using cDNA generated by reverse transcription from RNA from the sample as a template. The method of detecting variation according to claim 1, wherein, in the step (C), a change in a signal caused by a rise in temperature is measured by heating a hybrid body of the DNA and the detection probe. The method according to claim 1, wherein the step (B) and the step (C) are performed in parallel, wherein the temperature of the sample is lowered to form the hybrid formation, and the variation of the signal according to the temperature drop is measured. The method of detecting a mutation. A detection probe kit for use in the method for detecting a mutation according to claim 1, wherein the detection probe kit comprises a polynucleotide complementary to a detection target sequence and an inhibition for inhibiting hybridization of the detection probe to a non-detection sequence. For polynucleotides, The detection target sequence is a detection target DNA or a partial sequence thereof in which a detection site is mutated, and includes the detected detection site. The non-detection target sequence is the non-detection DNA or a partial sequence of the non-mutated detection site, the detection probe kit comprising the undetected detection site. The detection probe kit of claim 23, wherein the detection probe is a probe labeled with a labeling substance. 24. The inhibitory polynucleotide of claim 23, wherein the inhibitory polynucleotide that inhibits hybridization of the detection probe complementary to the detection target sequence and the detection probe to the non-detection sequence is identical except for a paired site with the detection site. Probe kit for detection of the sequence. The detection probe kit of claim 23, wherein the mutation is a mutation of the abl1 gene. 24. The method of claim 23, wherein the mutation is a nucleotide sequence of SEQ ID NO: 1 from 756 base G to C, the detection probe is a probe consisting of the nucleotide sequence of SEQ ID NO: 2, the detection to the non-detection target sequence A probe kit for detection, wherein the inhibitory polynucleotide that inhibits hybridization of the probe is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3. 24. The method of claim 23, wherein the mutation is a nucleotide of SEQ ID NO: 1 to the 758th base A to T, the detection probe is a probe consisting of the nucleotide sequence of SEQ ID NO: 4, the detection to the non-detection target sequence A probe kit for detection, wherein the inhibitory polynucleotide that inhibits the hybridization of the probe is a polynucleotide consisting of at least one nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 6. The method according to claim 23, wherein the mutation is a nucleotide sequence of SEQ ID NO: 1 to the 763th base G of A, the detection probe is a probe consisting of the nucleotide sequence of SEQ ID NO: 7, detection to the non-detection target sequence The detection probe kit of claim 1, wherein the inhibitory polynucleotide that inhibits hybridization of the probe is a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 8. As a method of interpreting data for determining the presence or absence of mutation of DNA, The method of interpreting data characterized by having the following steps (a) to (b): (a) calculating a Tm value from signal variation obtained in step (C) of the method for detecting variation described in claim 1, and (b) A step of determining the presence or absence of DNA variation from the Tm value calculated in the calculation step. delete delete delete The method according to claim 1, wherein in the step (A), a polynucleotide for inhibition that inhibits hybridization of the detection probe and the detection probe to the non-detection target sequence is added to the sample containing the DNA, and the gene is amplified. And amplification of the DNA, and hybridization of the probe to the DNA amplification product amplified by the gene amplification method in the step (B).

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